Construction of pool of interfering nucleic acids covering entire RNA target sequence and related compositions

ABSTRACT

The present invention provides a PCR based high-throughput method for preparing full-sites siRNA polynucleotide pool, comprising: DNase I random digestion; Loop-1 phosphate linker ligation; single PCR amplification; a type III restriction/modification enzyme digestion; blunt ending; Loop-2 phosphate linker ligation; double primer PCR; FokI digestion and cloning into an siRNA expression vector. The present invention enables the use of a type III restriction/modification enzyme linkers mediated PCR method for high-throughput preparing an siRNA polynucleotide pool, in which the functional length of siRNAs can be controllably distributed from 19-23 bp, thus completely mimic the natural siRNA length diversity, specially suitable for RNAi therapeutic targets screening. The present invention overcomes the bottlenecks and drawbacks of conventional siRNA polynucleotide pool construction technologies.

RELATED APPLICATIONS

This claims priority to International Patent Application No.PCT/CN2008/001283 filed on Jul. 7, 2008, which claims priority toChinese Patent Application No. 200710024217.6, filed on Jul. 23, 2007,texts of both applications being incorporated herein in their entiretyby reference.

FIELD OF THE INVENTION

The present invention relates to methods and compositions forconstructing a pool of interfering nucleic acids (iNA) from a sample andthe selected polynucleotide pools produced thereby, more particularlyfor preparing an iNA polynucleotide pool using type IIIrestriction/modification enzymes and the corresponding linkers.

BACKGROUND

The teachings of all of the references cited herein are incorporated intheir entirety by reference. An understanding of the biological role ofany gene comes only after observing the phenotypic consequences ofaltering the function of that gene in a living cell or organism. RNAinterference (RNAi) is a well-established experimental technology forsilencing gene expression both in cultured eukaryotic cells and livingorganisms. RNAi also be used as gene therapy for treating viralinfections, cancer, vascular diseases and other diseases in which thedown-regulation of a polypeptide would ameliorate the disease. RNAiinduces the sequence-specific degradation of a single mRNA species byshort interfering RNA (siRNA, a double-stranded small interference RNA),which is believed to be processed through the highly conserved Dicerfamily of RNase III enzymes in vivo. The process includes: 1) thedelivery of homologous double-stranded RNAs (dsRNAs) to the cytoplasm ofa cell. 2) dsRNA cleavage by the RNase III-like enzyme, Dicer, to 21˜23bp siRNAs. 3) siRNA incorporation into a protein complex, theRNA-induced silence complex (RISC). 4) the antisense strand of theduplex siRNA guiding the RISC to the homologous mRNA, where theRISC-associated endoribonuclease cleaves the target mRNA, resulting insilencing of the target gene.

As used herein a siRNA is also an interfering nucleic acid (iNA) as someof these interfering nucleic acids may contain a deoxyribonucleic acidplaced in the iNA to inhibit RNA nucleases. The siRNA molecule ofinterest can be synthesized in vitro by chemical and enzymatic (forexample by using the enzyme Dicer) methods. They can also be synthesizedin vivo. When a synthetic oligonucleotide is cloned into siRNAexpression vectors with a RNA polymerase III promoter (including U6,human H1, and tRNA promoters), or a polymerase II promoter with aminimal poly(A) signal sequence, siRNA can be transcribed in vivo.Typically a single promoter is used to express a short hairpin (shRNA)sequence, although two tandem polymerase III promoters have also beenused to transcribe the sense and antisense siRNA sequences. In additionto plasmid-based systems, PCR-derived siRNA expression cassettes basedon the single-promoter system is an alternative format for suppressingtransfected gene activity.

The siRNA-mediated gene silencing efficiency is affected by manyparameters. An important limiting fact is that only about 25% ofselected target siRNA sequences are functional due to some factors, suchas secondary structures, non-gene-specific reactions and other unknownfactors. Thus several synthetic siRNAs need to be generated and testedfor every target gene. Thus, it is very expensive and time consuming toidentify a suitable iNA construct. Another difficulty is the fact thatan iNA sequence may have enough complementarity to the sequence of asecond, unintended RNA. This is called the so-called off-target effect.Furthermore, to find the best siRNA binding sites (RNAi drug targets) isvery challenging. To resolve the off-target phenomenon, extensivestudies have been done on selecting specific target sequences for siRNA.Using algorithms based on sequence-efficacy correlations is the currentpractice for designing effective siRNAs. Although these criteriasignificantly increase chance of success for achieving gene silencing,there are many highly effective siRNA sequences that are not determinedby the current algorithms because different genes have differentsequence preferences. To ensure that the best iNAs are identified, ansiRNA library constructed from cDNA or DNA offers a better alternativeway to search for sequences that have the best potential silencingeffect. Moreover, such an siRNA library can be a useful research tool infunctional genomics, and useful for screening RNAi therapeutic targetsin a high-throughput manner.

Recently siRNA library approaches (such as whole genomic orgene-specific or domain-specific siRNA libraries) have become a powerfultool for screening RNAi therapeutic targets. In all those approaches,efforts to generate siRNA sequences having an appropriate length haveused MmeI-a type II restriction enzyme, by which a maximum 20nucleotides can be generated. However, a siRNA having a maximum lengthof 20 bp cannot completely mimic the cleavage product, having a lengthof 21-23 bp produced by an RNase III-like enzyme-Dicer. This can resultin the best siRNA target sites being underrepresented. In all theseapproaches, the double-stranded (ds) cDNA is randomly cleaved into smallfragments by DNase I (some use restriction enzymes for fragmentation butthe representation is an issue) and subsequently ligated to anartificial loop-anchor which contains a MmeI-type II restriction site,then digested by the MmeI restriction enzyme to cut 18-20 nucleotidesaway from the recognized site. Through complex and multiple processsteps including a second anchor ligation, loop extension and PAGEpurifications, the ds-cDNA is then converted into a 20-nt palindromicstructure with a loop (shRNA) and finally cloned into an siRNAexpression vector with an RNA polymerase III promoter. In all theseapproaches, efforts to generate siRNA sequences having an appropriatelength have used a MmeI restriction enzyme, by which only an iNA havinga maximum 20 nucleotides can be generated. This is the longest iNA thatcan be generated using the type II restriction enzymes.

However, there are a number drawbacks in this approach include thefollowing

1) An shRNA library cannot be generated by PCR due to a palindromicstructure. The complicated steps together with heavy cDNA loss inmultiple process steps make this approach difficult and impossible to bedeveloped into a high-throughput tool for functional genomics and forsiRNA therapeutic target screening.2) A palindromic structure is unstable during cloning in E. coli. Thiscan lead to reduction in library complexity and potential loss of thebest therapeutic target sites.3) An iNA having a maximum of 20 bp cannot completely mimic the cleavageproducts having 21 to 23 bp produced by an RNase III enzyme like Dicer.

An attempt to construct a siRNA library from cDNA using PCR hascurrently been reported. In this system, the dsRNAs corresponding to thecDNA of interest are prepared by T7 RNA polymerase mediatedtranscription from DNA templates flanked by a T7 RNA promoter andsubsequent annealing. The dsRNAs are then digested with cloned humanDicer in vitro, yielding 21˜23 bp siRNAs. A modified bacterial RNase IIIcan be used to replace Dicer, but the generated siRNA is 20˜25 bp.Cleavage products are denatured, purified by PAGE and dephosphorylated.RNA adapters are attached subsequently to the 3′- and 5′-ends of thecleavage products by T4 RNA ligase. RNAs are subsequently converted intodsDNA by RT-PCR using primers complementary to the adapters. Afterdigestion with appropriate restriction enzymes, the 21˜23 bp siRNAscorresponding to the cDNA fragments are ligated into an siRNA expressionvector having the dual RNA polymerase III promoters, U6 and H1. For adescription of the U6 and H1 promoters see US patent applicationpublication no. 20050064489. Taking advantage of PCR, this approach cantolerate the a heavy loss of starting material due to multiple processsteps and still generate enough molecules for cloning. Another advantageis that by using RNA fragmentation with Dicer, a distinct random poolsof iNAs having 21˜23 bp in length can be generated. However, thecDNA-RNA-cDNA conversion process steps are obviously a complex, and evenmore complicated than shRNA library construction described above.Furthermore, RNA degradation during multiple process steps (e.g., T7 DNApolymerase-mediated DNA to RNA transcription, Dicer digestion, RNA PAGEpurification, dephosphate and anchor ligation as well as RT-PCR) isunavoidable, which may result in the loss of some of the best siRNAtarget sites.

Another attempt at siRNA library construction is based on DNase Idigestion. In this approach, dscDNA is partially digested with DNase I,followed by PAGE gel purification isolating DNA fragments that are 20-30bp in lengths. These fragments are either directly blunt-end cloned intosiRNA expression vector or attached to a PCR anchor by ligation,followed by PCR amplification and subsequently cloning into siRNAexpression vector. It sounds much simpler and straightforward. However,cutting a nucleic acid fragment that is 20˜30 bp length from a PAGE gelis very challenging. Contamination with smaller nucleic acid fragmentsthat have a length of less than 16 bp and with larger nucleic acidfragments having a length greater than 30 bp cannot be avoided. The iNAsthat are too short having a length of less than 16 bp results in iNAsthat do not efficiently downregulate the target RNA. An iNA that has alength that is greater than 30 bp cannot be transfected into mammaliancells because their introduction into the mammalian cells activates aninterferon and protein kinase R (PKR) pathways in the cells, resultingin nonspecific gene silencing and apoptosis. Such an siRNA library maycontain a high frequency of undesirable (“junk”) clones which may notonly drastically impair the overall efficiency of the approach, but alsoseriously compromise the integrity of the data that are generated. Thus,this approach is not ideal for screening for the best siRNA sequencesite for functional genomics and RNAi therapeutics.

An ideal iNA library, especially a gene-specific library, should containevery site represented by multiple overlapping sequences, and individualsequences should have the widely accepted rational length of 19-23 bp,and should easily and simply be amplified by PCR to meet ahigh-throughput library construction format, accelerating the screeningsfor the best siRNA sequence site for functional genomics and RNAitherapeutics. Thus, there is a need to provide for a method for toproduce a library or pool iNA constructs having a length of 19-23 bps,which can be produced in a high-throughput manner, which covers thetarget sequence of an RNA.

DESCRIPTION

An object of the present invention is to provide a type IIIrestriction/modification enzyme mediated PCR high-throughput method forpreparing an iNA pool from a DNA sample (cDNA or genomic DNA and so on)for RNAi therapeutic targets screening. The resulting siRNApolynucleotide pool has iNAs constructing ranging in length from 16 to27 bp, more preferably, from 21˜23 bp. The pool of iNA constructscompletely mimics the length of siRNA naturally generated by Dicerenzyme in living cells. This overcomes the under-representation of allpossible iNA constructs produced by conventional siRNA polynucleotidepool construction approaches, which are mediated by a type IIrestriction enzyme-MmeI, in which the longest siRNA generated is 18-20bp in length.

The type III restriction enzyme-EcoP15I can cleave maximal nucleic acidfragment having a length of 25-27 bp of DNA outside of their recognitionsite. Currently no one uses any type III restriction/modificationenzymes for siRNA library construction mainly due to two technicaldifficulties: a.) the cleavage product having 25-27 bp is the ideallength for an iNA of 19-23 bp which is a widely accepted rational lengthof an siRNA; b.) two inversely oriented recognition sites of type IIIrestriction/modification enzymes is required for effective cleavage(using EcoP15I as an example):

Another object of the present invention is to provide an artificialoligonucleotide shaped in a loop after self-annealing (Loop-1 linkers)containing the recognition sites of a type III restriction/modificationenzyme and a type II restriction enzyme. The general structure formulaof Loop-1 linker(s) is:

(SEQ ID NO: 5) 5′ CTTTTN Type IIIase site N Type IIase site-PCRanchor-loop (SEQ ID NO: 6) 3′ GAAAAN Type IIIase site N Type IIasesite-PCR anchor-loop

Where:

In SEQ ID NO: 5, there can be 0 to 20 nucleotides between the ‘C’ andthe first ‘T’ at the 5′ end at the 3′ end there can be an additionalnucleotides ranging from 0 to 20 after the 3′ N;In SEQ ID NO: 6, there can be 0 to 20 nucleotides between the ‘G’ andthe first ‘T’ at the 3′ end, and 0 to 20 nucleotides after the A at the5′ end;N is any nucleotide bases preferably G, C, T, or A;Type IIase: type III restriction/modification enzyme;Type IIase: type II restriction enzyme.

Another object of the present invention is to provide another artificialoligonucleotide shaped in a loop after self-annealing (Loop-2 linker)containing a type II restriction enzyme recognition sequences.

The general structure formula of Loop-2 linker is:

(SEQ ID NO: 5)

5′CTTTTN Type IIase site-PCR anchor-loop having 0-20 nucleotides between‘C’ and first ‘T’ and 0-20 nucleotides after the 5′ ‘T’

(SEQ ID NO: 6)

3′ GAAAAN Type IIase site-PCR anchor-loop having 0-20 nucleotidesbetween ‘G’ and the first ‘A’ and 0-20 nucleotides after the final 3′‘A’.

Where

N is a nucleotide base preferably G, C, T, or A; andType IIase is a type II restriction enzyme.

Another object of the present invention is to provide a protocol for aPCR based high-throughput method for preparing an siRNA polynucleotidepool from a DNA sample, comprised of:

a.) Partially digesting cDNA or genomic DNA with DNaseI in the presenceof Mn²⁺ producing DNA constructs that are blunt-ended;

b.) Ligating Loop-1 linker(s) to one end of the blunted-ended DNAconstructs;

c.) Amplifying by PCR the DNA constructs with a single primer that is aportion of homologous sequences to the antisense strand of the Loop-1linker(s) (the strand with a poly A stretch) producing DNA constructscontaining double type III restriction/modification enzyme sites (e.g.,EcoP15I) in inversed orientation at the DNA both ends, which arecleavable; and

d.) Cleaving the DNA constructs with a type III restriction/modificationenzyme (e.g., EcoP15I). The enzyme cleaves a maximal 25-27 bp of DNAoutside of their recognition sites. The resulting siRNA polynucleotidepool distributed from 19 to 23 bp in length by adjusting the adjacentnumber of poly (A/T) sequences in the Loop-1 linker(s);

e.) Blunt-ending by filling in with a DNA polymerase in the present ofdNTPs;

f.) Loop-2 linker ligation. A type II restriction site (e.g., Fok1) isincluded in the Loop-2;

g.) Second PCR amplification with 5′ Loop-1 and 3′ Loop-2 primers;

h.) A type II restriction enzyme (e.g., FokI in both loops) digestion togenerate over polyA (A₄) sticky ends (cohesive ends) at both 5′ ends;

i.) Inserting the type II restriction enzyme digested constructs to apre-prepared siRNA expression vector with polyT (especially T₄) stickyor cohesive ends at both 3′ ends, flanked by two tandem RNA polymeraseIII promoters such as U6 and H1, to complete an siRNA polynucleotidepool construction. Ploy (A/T)₅ act as the initial and terminationsignals for RNA polymerase III promoters after cloning.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 A Schematic Diagram of Technical Flow-Chart and the Structure ofLoop-1 Linker(s) (using EcoP15I as an example):

FIG. 2 Schematic Diagram of an siRNA Expression Vector (pU6H1-GFP) Mapand MCS Sequences Before and After Cloning.

DEFINITIONS

Definitions of technical terms provided herein should be construed toinclude without recitation those meanings associated with these termsknown to those skilled in the art, and are not intended to limit thescope of the invention.

The use herein of the terms “a,” “an,” “the,” and similar terms indescribing the invention, and in the claims, are to be construed toinclude both the singular and the plural. The terms “comprising,”“having,” “including,” and “containing” are to be construed asopen-ended terms which mean, for example, “including, but not limitedto.”

Recitation of a range of values herein refers individually to each andany separate value falling within the range as if it were individuallyrecited herein, whether or not some of the values within the range areexpressly recited. Specific values employed herein will be understood asexemplary and not to limit the scope of the invention.

As used herein, the term interfering nucleic acid (iNA) refers to anucleic acid duplexes having a sense and antisense strand, which whenentered into a RISC complex induces enzymatic degradation of mRNA.Generally each strand contains predominantly RNA nucleotides but thestrands can contain RNA analogs, RNA and RNA analogs, RNA and DNA, RNAanalogs and DNA, or one strand that is completely DNA and one strandthat is RNA as long as the iNA construct induces enzymatic degradationof a homologous mRNA.

The term “short interfering nucleic acid”, “iNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PC7Publication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCFPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). For example the iNA can bea double-stranded polynucleotide molecule comprising self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof.

The iNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary (i.e.each strand comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 15 to about 30,e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 base pairs; the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof (e.g., about 15 to about 25 or more nucleotides of the iNAmolecule are complementary to the target nucleic acid or a portionthereof). Alternatively, the iNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the iNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s).

The iNA can be a polynucleotide with a duplex, asymmetric duplex,hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a separate target nucleic acid molecule or a portion thereofand the sense region having nucleotide sequence corresponding to thetarget nucleic acid sequence or a portion thereof. The iNA can be acircular single-stranded polynucleotide having two or more loopstructures and a stem comprising self-complementary sense and antisenseregions, wherein the antisense region comprises nucleotide sequence thatis complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof, and wherein the circular polynucleotide can be processed eitherin vivo or in vitro to generate an active iNA molecule capable ofmediating RNAi. The iNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (forexample, where such iNA molecule does not require the presence withinthe iNA molecule of nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell., 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or5′,3′-diphosphate. In certain embodiments, the iNA molecule of theinvention comprises separate sense and antisense sequences or regions,wherein the sense and antisense regions are covalently linked bynucleotide or non-nucleotide linkers molecules as is known in the art,or are alternately non-covalently linked by ionic interactions, hydrogenbonding, van der waals interactions, hydrophobic interactions, and/orstacking interactions. In certain embodiments, the iNA molecules of theinvention comprise nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, the iNAmolecule of the invention interacts with nucleotide sequence of a targetgene in a manner that causes inhibition of expression of the targetgene.

As used herein, iNA molecules need not be limited to those moleculescontaining only RNA, but further encompasses chemically modifiednucleotides and non-nucleotides. In certain embodiments, the shortinterfering nucleic acid molecules of the invention lack 2′-hydroxy(2′-OH) containing nucleotides. Applicant describes in certainembodiments short interfering nucleic acids that do not require thepresence of nucleotides having a 2′-hydroxy group for mediating RNAi andas such, short interfering nucleic acid molecules of the inventionoptionally do not include any ribonucleotides (e.g., nucleotides havinga 2′-OH group). Such iNA molecules that do not require the presence ofribonucleotides within the iNA molecule to support RNAi can however havean attached linker or linkers or other attached or associated groups,moieties, or chains containing one or more nucleotides with 2′-OHgroups. Optionally, iNA molecules can comprise ribonucleotides at about5, 10, 20, 30, 40, or 50% of the nucleotide positions. The modifiedshort interfering nucleic acid molecules of the invention can also bereferred to as short interfering modified oligonucleotides “siMON.” Asused herein, the term iNA is meant to be equivalent to other terms usedto describe nucleic acid molecules that are capable of mediatingsequence specific RNAi, for example short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid, short interfering modified oligonucleotide, chemically-modifiedsiRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost-transcriptional gene silencing, translational inhibition, orepigenetics. For example, iNA molecules of the invention can be used toepigenetically silence genes at both the post-transcriptional level orthe pre-transcriptional level. In a non-limiting example, epigeneticregulation of gene expression by iNA molecules of the invention canresult from iNA mediated modification of chromatin structure ormethylation pattern to alter gene expression (see, for example, Verdelet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237).

As used herein, the term “iNA duplex” is a generic term used throughoutthe specification to include interfering nucleic acids (iNAs), hairpiniNAs which can be cleaved in vivo to form iNAs. The iNA duplexes hereinalso include expression vectors (also referred to as iNA expressionvectors) capable of giving rise to transcripts that form iNA duplexes orhairpin iNAs in cells, and/or transcripts, which can produce iNAs invivo. Optionally, the iNA include single strands that form a duplex by ahairpin-loop or double strands of iNA. The iNA is a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence in a targetribonucleic acid molecule for down regulating expression, or a portionthereof. The sense strand or antisense strand have one or more nicks ornucleotide. The terminal structure of iNA may be either blunt orcohesive (overhanging) as long as the iNA can silence the target mRNA.The cohesive (overhanging) end structure is not limited only to the 3′overhang, as the 5′overhanging structure may be included as long as itis capable of inducing the RNAi effect. In addition, the number ofoverhanging nucleotides is not limited to the reported 2 or 3, but canbe any number as long as the overhang is capable of inducing the RNAieffect. For example, the overhang may be 1 to 8, or 2 to 4 nucleotides.

As used herein the length of the iNA duplex is determined by countingthe number of nucleotides in the duplex starting at the first base-pairat the 5′ end of the sense strand and ending at the last base-pair atthe 3′ end of the sense strand.

In genetics, microRNAs (miRNA) are single-stranded RNA molecules ofabout 21-23 nucleotides in length, which regulate gene expression.miRNAs are encoded by genes that are transcribed from DNA but nottranslated into protein (non-coding RNA); instead they are processedfrom primary transcripts known as pri-miRNA to short stem-loopstructures called pre-miRNA and finally to functional miRNA. MaturemiRNA molecules are partially complementary to one or more messenger RNA(mRNA) molecules, and their main function is to downregulate geneexpression.

Modified nucleotides in an iNA molecule can be in the antisense strand,the sense strand, or both. For example, modified nucleotides can have aNorthern conformation (e.g., Northern pseudorotation cycle, see, forexample, Saenger, Principles of Nucleic Acid Structure, Springer-Verlaged., 1984). Examples of nucleotides having a Northern configurationinclude locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethoxy (MOE)nucleotides, 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methylnucleotides. Chemically modified nucleotides can be resistant tonuclease degradation while at the same time maintaining the capacity tomediate RNAi. A conjugate molecule attached to a chemically-modified iNAmolecule is a polyethylene glycol, human serum albumin, or a ligand fora cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified iNA molecules are described inVargeese, et al., U.S. Patent Publication No. 20030130186 and U.S.Patent Publication No. 20040110296, which are each hereby incorporatedby reference in their entirety.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications. For areview see Usman and Cedergren, TIBS 17:34, 1992; Usman, et al, NucleicAcids Symp. Ser. 31:163, 1994; Burgin, et al, Biochemistry 35:14090,1996. Sugar modification of nucleic acid molecules have been extensivelydescribed in the art. See Eckstein et al., International Publication PCTNo. WO 92/07065; Perrault, et al. Nature 344:565-568, 1990; Pieken, etal. Science 253:314-317, 1991; Usman and Cedergren, Trends in Biochem.Sci. 17:334-339, 1992; Usman et al. International Publication PCT No. WO93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman, et al., J.Biol. Chem. 270:25702, 1995; Beigelman, et al., International PCTPublication No. WO 97/26270; Beigelman, et al., U.S. Pat. No. 5,716,824;Usman, et al., U.S. Pat. No. 5,627,053; Woolf, et al., International PCTPublication No. WO 98/13526; Thompson, et al., Karpeisky, et al,Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (NucleicAcid Sciences) 48:39-55, 1998; Verma and Eckstein, Annu. Rev. Biochem.67:99-134, 1998; and Burlina, et al., Bioorg. Med. Chem. 5:1999-2010,1997. Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into nucleic acid molecules withoutmodulating catalysis. In view of such teachings, similar modificationscan be used as described herein to modify the iNA nucleic acid moleculesof the claimed duplexes so long as the ability of iNA to promote RNAi incells is not significantly inhibited.

The iNA duplexes may contain modified iNA molecules, with phosphatebackbone modifications comprising one or more phosphorothioate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl,substitutions. For a review of oligonucleotide backbone modifications,see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 1995, pp. 331-417, andMesmaeker, et al., “Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research,” ACS, 1994, pp. 24-39.Examples of chemical modifications that can be made in an iNA includephosphorothioate internucleotide linkages, 2′-deoxyribonucleotides,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation. The antisense region of a iNA molecule can include aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. The antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. The 3′-terminal nucleotide overhangs of a iNA moleculecan include ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. The3′-terminal nucleotide overhangs can include one or more universal baseribonucleotides. The 3′-terminal nucleotide overhangs can comprise oneor more acyclic nucleotides. For example, a chemically-modified iNA canhave 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotidelinkages in one strand, or can have 1 to 8 or more phosphorothioateinternucleotide linkages in each strand. The phosphorothioateinternucleotide linkages can be present in one or both oligonucleotidestrands of the iNA duplex, for example in the sense strand, theantisense strand, or both strands. In some embodiments, a iNA moleculeincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more purine phosphorothioateinternucleotide linkages in the sense strand, the antisense strand, orin both strands.

The iNA molecules, which can be chemically-modified, can be synthesizedby: (a) synthesis of two complementary strands of the iNA molecule; and(b) annealing the two complementary strands together under conditionssuitable to obtain a double-stranded iNA molecule. In some embodiments,synthesis of the complementary portions of the iNA molecule is by solidphase oligonucleotide synthesis, or by solid phase tandemoligonucleotide synthesis.

The term “nucleotide” as used herein, refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a phosphorylated sugar.Nucleotides are recognized in the art to include natural bases(standard), and modified bases well known in the art. Such bases aregenerally located at the 1′ position of a nucleotide sugar moiety.Nucleotides generally comprise a base, sugar and a phosphate group. Thenucleotides can be unmodified or modified at the sugar, phosphate and/orbase moiety, (also referred to interchangeably as nucleotide analogs,modified nucleotides, non-natural nucleotides, non-standard nucleotidesand other; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of chemically modified and other natural nucleicacid bases that can be introduced into nucleic acids include, forexample, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra).

By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents; such bases can be used at any position, for example, withinthe catalytic core of an enzymatic nucleic acid molecule and/or in thesubstrate-binding regions of the nucleic acid molecule. Nucleotides areorganic compounds comprised of three joined structures: a nitrogenousbase, a sugar, and a phosphate group. The most common nucleotides can bedivided into two groups (purines and pyrimidines) based on the structureof the nitrogenous base. The joined sugar is either ribose ordeoxyribose. A nucleotide is composed of a ring of nitrogen, carbon andoxygen atoms, a five-carbon sugar (together referred to as a nucleoside)and one phosphate group. The name of the nucleotide is determined by itsbase. For example an adenine (A) nucleotide has an adenine base, aguanine (G) nucleotide has a guanine base, a thymine (T) nucleotide hasa thymine base, a uracil (U) nucleotide has a uracil base, and acytosine (C) nucleotide has a cytosine base.

A strand of DNA contains nucleotides and a DNA molecule is made up of 2polynucleotide chains arranged on the double helix (the backbone). Thesenucleotides are composed of three parts: a phosphate, a sugar(deoxyribose), and a type of compound base. The deoxyribose andphosphate form the backbone of nucleic acid (the side of the ladder)while the base connect the two polynucleotide chains (like the rungs ofthe ladder). There are four main types of bases, adenine, guanine,thymine and cytosine but they are just referred to by the first letterin their name, A, G, T and C respectively.

Ribonucleic acid (RNA) is a nucleic acid that is comprised of a longchain of nucleotide units. Each nucleotide consists of a nitrogenousbase, a ribose sugar, and a phosphate. RNA is very similar to DNA, butdiffers in a few important structural details: in the cell, RNA isusually single-stranded, while DNA is usually double-stranded; RNAnucleotides contain ribose while DNA contains dexoyribose (a type ofribose that lacks one oxygen atom); and RNA has the base uracil ratherthan thymine that is present in DNA. RNA is transcribed from DNA byenzymes called RNA polymerases and is generally further processed byother enzymes.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers, etal, Methods in Enzymology 211:3-19, 1992; Thompson, et al.,International PCT Publication No. WO 99/54459; Wincott, et al., NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al., Methods Mol. Bio. 74:59,1997; Brennan, et al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan,U.S. Pat. No. 6,001,311. Synthesis of RNA, including certain iNAmolecules of the invention, follows general procedures as described, forexample, in Usman, et al., J. Am. Chem. Soc. 109:7845, 1987; Scaringe,et al., Nucleic Acids Res. 18:5433, 1990; and Wincott, et al, NucleicAcids Res. 23:2677-2684, 1995; Wincott, et al, Methods Mol. Bio. 74:59,1997. The double-stranded structure may be formed by self-complementaryiNA strand such as occurs for a hairpin RNA or by annealing of twodistinct complementary iNA strands.

“Overlapping” refers to when two iNA fragments have sequences whichoverlap by a plurality of nucleotides on one strand, for example, wherethe plurality of nucleotides (nt) numbers as few as 2-5 nucleotides orby 5-10 nucleotides or more.

“One or more iNAs” refers to iNAs that differ from each other on thebasis of primary sequence.

By “target site” or “target sequence” or “targeted sequence” is meant asequence within a target nucleic acid (e.g., RNA) that is “targeted” forcleavage mediated by an iNA duplex which contains sequences within itsantisense region that are complementary to the target sequence.

A hybrid iNA molecule is an iNA that is a double-stranded nucleic acid.Instead of a double-stranded RNA molecule, a hybrid iNA is comprised ofan RNA strand and a DNA strand. Preferably, the RNA strand is theantisense strand as that is the strand that binds to the target mRNA.The hybrid iNA created by the hybridization of the DNA and RNA strandshave a hybridized complementary portion and preferably at least one3′overhanging end.

To “modulate gene expression” as used herein is to up-regulate ordown-regulate expression of a target gene, which can includeupregulation or down-regulation of mRNA levels present in a cell, or ofmRNA translation, or of synthesis of protein or protein subunits,encoded by the target gene.

The terms “inhibit,” “down-regulate,” or “reduce expression,” as usedherein mean that the expression of the gene, or level of RNA moleculesor equivalent RNA molecules encoding one or more proteins or proteinsubunits, or level or activity of one or more proteins or proteinsubunits encoded by a target gene, is reduced below that observed in theabsence of the nucleic acid molecules (e.g., iNA) of the invention.

“Gene silencing” as used herein refers to partial or complete inhibitionof gene expression in a cell and may also be referred to as “geneknockdown.” The extent of gene silencing may be determined by methodsknown in the art, some of which are summarized in InternationalPublication No. WO 99/32619.

“Restriction Enzyme” A restriction enzyme (or restriction endonuclease)is an enzyme that cuts double-stranded DNA at specific recognitionnucleotide sequences known as restriction sites. Such enzymes, found inbacteria and archaea, are thought to have evolved to provide a defensemechanism against invading viruses. To cut the DNA, a restriction enzymemakes two incisions, once through each sugar-phosphate backbone (i.e.each strand) of the DNA double helix. Restriction endonucleases arecategorized into three general groups (Types I, II and III) based ontheir composition and enzyme cofactor requirements, the nature of theirtarget sequence, and the position of their DNA cleavage site relative tothe target sequence.

The term “complementarity” refers to the ability of a nucleic acid toform hydrogen bond(s) with another RNA sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., RNA interference, enzymatic nucleic acid cleavage,antisense or triple helix inhibition. Determination of binding freeenergies for nucleic acid molecules is well known in the art (see, e.g.,Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Frier etal., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987,J. Am. Chem. Soc. 109:3783-3785). A percent complementarity indicatesthe percentage of contiguous residues in a nucleic acid molecule whichcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). “Perfectly complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “ribonucleotide” or “2′-OH” is meant a nucleotide with a hydroxylgroup at the 2′ position of a β-D-ribo-furanose moiety.

Joining linear DNA fragments together with covalent bonds is calledligation or ligating the fragments together. More specifically, DNAligation involves creating a phosphodiester bond between the 3′ hydroxylof one nucleotide and the 5′ phosphate of another. The enzyme used toligate DNA fragments is T4 DNA ligase, which originates from the T4bacteriophage. This enzyme will ligate DNA fragments having overhanging,cohesive ends that are annealed together, as in the EcoRI examplebelow—this is equivalent to repairing “nicks” in duplex DNA. T4 DNAligase will also ligate fragments with blunt ends, although higherconcentrations of the enzyme are usually recommended for this purpose.

A restriction digest is a procedure used in molecular biology to prepareDNA for analysis or other processing. It is also known as DNAfragmentation. It uses a number of restriction enzymes to selectivelycleave strands of DNA into shorter fragments, or to isolate shortfragments of interest. The resulting digested DNA is very oftenselectively amplified using PCR, making it more suitable for analyticaltechniques such as agarose gel electrophoresis, and chromatography. Itis used in genetic fingerprinting, and RFLP analysis.

A given restriction enzyme cuts DNA segments within a specificnucleotide sequence. These recognition sequences are typically four,six, eight, ten, or twelve nucleotides long. Because there are only somany ways to arrange the four nucleotides which compose DNA (Adenine,Thymine, Guanine and Cytosine) into a four- to twelve-nucleotidesequence, recognition sequences tend to occur by chance in any longsequence. Restriction enzymes specific to hundreds of distinct sequenceshave been identified and synthesized for sale to laboratories, and as aresult, several potential “restriction sites” appear in almost any geneor locus of interest on any chromosome. Furthermore, almost allartificial plasmids include an (often entirely synthetic) polylinder(also called “multiple cloning site”) that contains dozens ofrestriction enzyme recognition sequences within a very short segment ofDNA. This allows the insertion of almost any specific fragment of DNAinto plasmid vectors, which can be efficiently “cloned” by insertioninto replicating bacterial cells.

After restriction digest, DNA can then be analysed using gelelectrophoresis. In gel electrophoresis, a sample of DNA is first“loaded” onto a slab of agarose gel (literally pipetted into small wellsat one end of the slab). The gel is then subjected to an electric field,which draws the negatively charged DNA across it. The molecules travelat different rates (and therefore stop at different distances) dependingon their net charge (more highly charged particles travel further), andsize (smaller particles travel further). Since none of the fournucleotide bases carry any charge, net charge becomes insignificant andsize is the main factor affecting rate of diffusion through the gel. Netcharge in DNA is produced by the sugar-phosphate backbone.

Polymerase chain reaction (PCR) is a technique widely used in molecularbiology. It derives its name from one of its key components, a DNApolymerase used to amplify a piece of DNA by in vitro enzymaticreplication. As PCR progresses, the DNA thus generated is itself used asa template for replication. This sets in motion a chain reaction inwhich the DNA template is exponentially amplified. With PCR it ispossible to amplify a single or few copies of a piece of DNA acrossseveral orders of magnitude, generating millions or more copies of theDNA piece. PCR can be extensively modified to perform a wide array ofgenetic manipulations.

PCR is very versatile. Many types of samples can be analyzed for nucleicacids. Most PCR uses DNA as a target, rather than RNA, because of thestability of the DNA molecule and the ease with which DNA can beisolated. Almost all PCR applications employ a heat-stable DNApolymerase, such as Taq polymerase, an enzyme originally isolated fromthe bacterium Thermus aquaticus. This DNA polymerase enzymaticallyassembles a new DNA strand from DNA building blocks, the nucleotides, byusing single-stranded DNA as a template and DNA oligonucleotides (alsocalled DNA primers), which are required for initiation of DNA synthesis.The vast majority of PCR methods use thermal cycling, i.e., alternatelyheating and cooling the PCR sample to a defined series of temperaturesteps.

These thermal cycling steps are necessary to physically separate thestrands (at high temperatures) in a DNA double helix (DNA melting) usedas template during DNA synthesis (at lower temperatures) by the DNApolymerase to selectively amplify the target DNA. The selectivity of PCRresults from the use of primers that are complementary to the DNA regiontargeted for amplification under specific thermal cycling conditions.PCR is used to amplify specific regions of a DNA strand (the DNAtarget). This can be a single gene, a part of a gene, or a non-codingsequence. Most PCR methods typically amplify DNA fragments of up to 10kilo base pairs (kb), although some techniques allow for amplificationof fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. Thesecomponents include:

-   -   DNA template that contains the DNA region (target) to be        amplified.    -   Two primers, which are complementary to the DNA regions at the        5′ (five prime) or 3′ (three prime) ends of the DNA region.    -   Taq polymerase or another DNA polymerase with a temperature        optimum at around 70° C.    -   Deoxynucleoside triphosphates (dNTPs), the building blocks from        which the DNA polymerases synthesizes a new DNA strand.    -   Buffer solution providing a suitable chemical environment for        optimum activity and stability of the DNA polymerase.    -   Divalent cations, magnesium or manganese ions; generally Mg²⁺ is        used, but Mn²⁺ can be utilized for PCR-mediated DNA mutagenesis,        as higher Mn²⁺ concentration increases the error rate during DNA        synthesis.    -   Monovalent cation potassium ions.

PCR is commonly carried out in a reaction volume of 10-200 μL in smallreaction tubes (0.2-0.5 mL volumes) in a thermal cycler. The thermalcycler heats and cools the reaction tubes to achieve the temperaturesrequired at each step of the reaction (see below). Many modern thermalcyclers make use of the Peletier effect which permits both heating andcooling of the block holding the PCR tubes simply by reversing theelectric current. Thin-walled reaction tubes permit favorable thermalconductivity to allow for rapid thermal equilibration. Most thermalcyclers have heated lids to prevent condensation at the top of thereaction tube. Older thermocyclers lacking a heated lid require a layerof oil on top of the reaction mixture or a ball of wax inside the tube.The PCR usually consists of a series of 20 to 40 repeated temperaturechanges called cycles; each cycle typically consists of 2-3 discretetemperature steps. Most commonly PCR is carried out with cycles thathave three temperature steps. The cycling is often preceded by a singletemperature step (called hold) at a high temperature (>90° C.), andfollowed by one hold at the end for final product extension or briefstorage. The temperatures used and the length of time they are appliedin each cycle depend on a variety of parameters. These include theenzyme used for DNA synthesis, the concentration of divalent ions anddNTPs in the reaction, and the melting temperature (Tm) of the primers.

Initialization step: This step consists of heating the reaction to atemperature of 94-96° C. (or 98° C. if extremely thermostablepolymerases are used), which is held for 1-9 minutes. It is onlyrequired for DNA polymerases that require heat activation by hot-startPCR.

Denaturation step: This step is the first regular cycling event andconsists of heating the reaction to 94-98° C. for 20-30 seconds. Itcauses melting of DNA template and primers by disrupting the hydrogenbonds between complementary bases of the DNA strands, yielding singlestrands of DNA.

Annealing step: The reaction temperature is lowered to 50-65° C. for20-40 seconds allowing annealing of the primers to the single-strandedDNA template. Typically the annealing temperature is about 3-5 degreesCelsius below the Tm of the primers used. Stable DNA-DNA hydrogen bondsare only formed when the primer sequence very closely matches thetemplate sequence. The polymerase binds to the primer-template hybridand begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on theDNA polymerase used; Taq polymerase has its optimum activity temperatureat 75-80° C., and commonly a temperature of 72° C. is used with thisenzyme. At this step the DNA polymerase synthesizes a new DNA strandcomplementary to the DNA template strand by adding dNTPs that arecomplementary to the template in 5′ to 3′ direction, condensing the5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end ofthe nascent (extending) DNA strand. The extension time depends both onthe DNA polymerase used and on the length of the DNA fragment to beamplified. As a rule-of-thumb, at its optimum temperature, the DNApolymerase will polymerize a thousand bases per minute. Under optimumconditions, i.e., if there are no limitations due to limiting substratesor reagents, at each extension step, the amount of DNA target isdoubled, leading to exponential (geometric) amplification of thespecific DNA fragment.

Final elongation: This single step is occasionally performed at atemperature of 70-74° C. for 5-15 minutes after the last PCR cycle toensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4-15° C. for an indefinite time may be employedfor short-term storage of the reaction.

To check whether the PCR generated the anticipated DNA fragment (alsosometimes referred to as the amplimer or amplicon), agarose gelelectrophoresis is employed for size separation of the PCR products. Thesize(s) of PCR products is determined by comparison with a DNA ladder (amolecular weight marker), which contains DNA fragments of known size,run on the gel alongside the PCR products. See Joseph Sambrook and DavidW. Russel (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press. Chapter 8: Invitro Amplification of DNA by the Polymerase Chain Reaction

Type I

Type I restriction enzymes were the first to be identified and arecharacteristic of two different strains (K-12 and B) of E. coli. Theseenzymes cut at a site that differs, and is some distance (at least 1000bp) away, from their recognition site. The recognition site isasymmetrical and is composed of two portions—one containing 3-4nucleotides, and another containing 4-5 nucleotides—separated by aspacer of about 6-8 nucleotides.

Type II

Typical type II restriction enzymes differ from type I restrictionenzymes in several ways. They are composed of only one subunit, theirrecognition sites are usually undivided and palindromic and 4-8nucleotides in length, they recognize and cleave DNA at the same site,and require only Mg²⁺ as a cofactor. There are subcategories based ondeviations from typical characteristics of type II enzymes. Thesesubgroups are defined using a letter suffix.

Type IIB restriction enzymes (e.g. BcgI and BplI) are multimerscontaining more than one subunit. They cleave DNA on both sides of theirrecognition to cut out the recognition site. They require both AdoMetand Mg²⁺ cofactors. Type IIE restriction endonucleases (e.g. NaeI)cleave DNA following interaction with two copies of their recognitionsequence. One recognition site acts as the target for cleavage, whilethe other acts as an allosteric effector that speeds up or improves theefficiency of enzyme cleavage. Similar to type IIE enzymes, type IIFrestriction endonucleases (e.g. NgoMIV) interact with two copies oftheir recognition sequence but cleave both sequences at the same time.Type IIG restriction endonucleases (Eco57I) do have a single subunit,like classical Type II restriction enzymes, but require the cofactorAdoMet to be active. Type IIM restriction endonucleases, such as DpnI,are able to recognize and cut methylated DNA. Type IIS restrictionendonucleases (e.g. FokI) cleave DNA at a defined distance from theirnon-palindromic asymmetric recognition sites.

These enzymes may function as dimers. Similarly, Type IIT restrictionenzymes (e.g., Bpu10I and BslI) are composed of two different subunits.Some recognize palindromic sequences while others have asymmetricrecognition sites.

Type III

Type III restriction enzymes (e.g. EcoP15 and EcoP151) recognize twoseparate non-palindromic sequences that are inversely oriented. They cutDNA about 20-30 base pairs after the recognition site. These enzymescontain more than one subunit and require AdoMet and ATP cofactors fortheir roles in DNA methylation and restriction, respectively.

“Recognition site” Restriction enzymes recognize a specific sequence ofnucleotides and produce a double-stranded cut in the DNA. Whilerecognition sequences vary widely, with lengths between 4 and 8nucleotides, many of them are palindromic; that is, the sequence on onestrand reads the same in the reverse direction on the complementarystrand. The meaning of “palindromic” in this context is different fromwhat one might expect from its linguistic usage: GTAATG (SEQ ID NO:68)is not a palindromic DNA sequence, but GTATAC (SEQ ID NO:69) is [GTATAC(SEQ ID NO:69 is complementary to CATATG (SEQ ID NO:70)].

Recognition sequences in DNA differ for each restriction enzyme,producing differences in the length, sequence and strand orientation (5′end or the 3′ end) of a sticky-end “overhang” of an enzyme restriction.

A vector is any vehicle used to transfer foreign genetic material intoanother cell. The vector itself is generally a DNA sequence thatconsists of an insert (transgene) and a larger sequence that serves asthe “backbone” of the vector. The purpose of a vector to transfergenetic information to another cell is typically to isolate, multiply,or express the insert in the target cell. Vectors called expressionvectors (expression constructs) specifically are for the expression ofthe transgene in the target cell, and generally have a promotersequencethat drives expression of the transgene. The expression may be of apolypeptide, protein or RNA.

An iNA expression vector is a vector that expresses an iNA.

A DNA construct is a DNA that is to be expressed when inserted into avector and transfected into a cell.

Insertion of a vector into the target cell is generally calledtransfection, although insertion of a viral vector is often calledtransduction.

In the Loop-1 linker(s) of present invention as described in FIG. 1 and“The general structure formula” described above, the poly(T/A) sequencesbetween EcoP15I and DNA inserts play three key roles:

a.) The resulting DNA insert length after EcoP15I cleavage can bemodified to have a length of from 19-23 bp by the addition or deletionof poly(T/A) sequences between EcoP15I in Loop-1 and DNA substrates. Ifone uses a Loop-1 linker only with a distinct poly(T/A) number, thelength of DNA insert can also be fixed at a certain length to constructan siRNA polynucleotide pool at a distinct length, or mix all Loop-1linkers together to generate an siRNA polynucleotide pool having a 19-23bp size distribution, according to the needs. Using this method an siRNApolynucleotide pool can be produced in which the lengths of the iNAconstructs can be varied by the addition or deletion of the A/Tsequences in Loop-1 linker(s) resulting in iNA pools being constructedin which the lengths of the constructs are 16-18 bp or 24-27 bp;

b.) Generating polyA (A₄) cohesive ends at both 5′ ends by FokI, a typeII restriction enzyme, conducting a poly T/A cloning. For theconstruction of a 24-27 bp iNA polynucleotide pool construction, othertype restriction enzymes can be employed to create the correspondingcloning sites;

c.) Generating the initiation and termination signals of RNA polymeraseIII promoters. After the iNA polynucleotide inserts are cloned into asiRNA expression vector, the “AAAAA” (SEQ ID NO: 7) (an initiationsignal) and “TTTTT” (SEQ ID NO: 8)(a termination signal) of thepromoters are generated. There are no additional cloning sequencesbetween RNA polymerase III (U6 and H1) promoters and DNA inserts, whichcan increase specificity of RNAi therapeutic targets screening.

The number of “N” between EcoP15I and FokI maintains the A₄ adhesiveends (over A₄ hanging ends at both 5′ ends). As soon as ligation into ansiRNA expression vector which has T5/A1 cohesive ends, the length ofinitiation signal [AAAAA (SEQ ID NO:7)] and termination signal [TTTTT(SEQ ID NO: 8)] for RNA polymerase III promoters (U6 and H1) arecreated.

Adding an additional “G” before the initiation signal “AAAAA” (SEQ IDNO: 7) of RNA polymerase III promoters improves an siRNA transcriptionefficiency. A feature of the present invention is that Loop-1 linkerscontain a “G” before Poly(A), except for construction of 23 bp siRNApolynucleotide pool. It is widely accepted that placing “G” before theinitial signal “AAAAA” (SEQ ID NO: 7) enhances RNA polymerase IIIpromoter activity, especially for the U6 promoter. However, the “G”needs to be omitted to maintain siRNA polynucleotide 23 bp in length byLoop-1 linker and can be added to an siRNA expression vector. If needed,for 24-27 bp siRNA polynucleotide pool construction, one should placethe initiation/termination signals together with its enhancing base “G”into an siRNA expression vector. In this case an appropriate cloningsite has to be selected in both Loop-1 linker(s) and vector to conductan efficient cloning. The present invention provides unlimited formats,especially with the future appearance of other type III restrictionenzymes that have longer cleavage outside of their recognition sequencesthan EcoP15I or EcoP15.

The PCR anchor and the loop structure designed in Loop-1 linker(s) areused to select a correct orientation for type IIIrestriction/modification enzyme cleavage. As mentioned above, the typeIII restriction/modification enzymes require two 5′ sequences ininversed orientations within the same DNA molecule to accomplishcleavage. Single primer PCR enables to select such molecules. For asuccessful single primer-based PCR selection, a loop shaped structure ishelpful. Unlike a tandem linker, a looped linker has no primer activity,thus ensures the single primer PCR successful.

In a preferred embodiment of the present invention there are thefollowing steps:

1) DNA is randomly and partially digested producing blunt-endedfragments by DNase I in the presence of Mn²⁺. DNase I, (RNase-free) isan endonuclease that nonspecifically cleaves DNA to produce productshaving 5′-phosphorylated and 3′-hydroxylated end. The concentration ofDNase I for the partial digestion is 0.01˜0.3 U of DNaseI per 1 g DNAdepending on the length and purity of DNA as well as the source of theenzyme. Due to a nonspecific fragmentation, the resulting products arerandom fragments that can be a representative distribution of the siRNApolynucleotide pool generated. In the presence of Mn²⁺ (a finalconcentration is 1 mM in a buffer, e.g., NEB buffer-2), the partiallydigested DNA are blunt-ended. The preferable length for downstreamLoop-1 linker(s) ligation and siRNA polynucleotide pool construction is100-300 bp.

2) Loop-1 Linker(s) ligation:

The present invention has designed various kinds of corresponding Loop-1linker(s). They can be used in a mixture to generate 19-, 20-, 21-, 22-,and 23 bp siRNA polynucleotide pool in length simultaneously, orseparately use if one has specially interest in a distinct lengthpolynucleotide of siRNA. FIG. 1 shows the general structure of a DNAconstruct of a Loop-1 linker having a EcoP151/Fok1 restriction sites.Examples of such Loop-1 constructs include:

For 19 bp iNA Library:            EcoP151 Fok1 5′ CTTTTTTTCTGCTG CATCC(SEQ ID NO: 79) 3′ GAAAAAAAGACGAC GTAGG (SEQ ID NO: 80) For 20 bp siRNALibrary:            EcoP151 Fok1 5′ CTTTTTTCTGCTGNCATCC (SEQ ID NO: 71)3′ GAAAAAAGACGACNGTAGG (SEQ ID NO: 72) For 21 bp siRNA Library:        EcoP151  Fok1 5′ CTTTTTCTGCTGNNCATCC (SEQ ID NO: 73)3′ GAAAAAGACGACNNGTAGG; (SEQ ID NO: 74) For 22 bp siRNA Library:        EcoP151  Fok1 5′ CTTTTCTGCTGNNNCATCC (SEQ ID NO: 75)3′ GAAAAGACGACNNNGTAGG; (SEQ ID NO: 76) For 23 bp siRNA Library:        EcoP151  Fok1 5′ TTTTCTGCTGNNNNCATCC (SEQ ID NO: 77)3′ AAAAGACGACNNNNGTAGG. (SEQ ID NO: 78)

As described in the Loop-1 phosphate linker(s) structure, a blunt-endligation with the partially digested DNA can be performed by any DNAligases, more preferably, a T4 DNA ligase in the presence of ATP. Themolar ratio of Loop-1 linker(s) and DNA is 10:1.

3) Single Primer PCR Amplification:

Efficient cleavage by type III restriction enzymes requires the presenceof two inversely oriented substrate sites as described above. A head tohead configuration in inverse orientation is required. Using EcoP15I asan Example:

Where there is a poly(A) sequence after the final N at the 3′ end of SEQID NO:1, a poly(T) sequence for the first nucleotide at the 5′ end ofSEQ ID NO:2, a poly(T) sequence after the last nucleotide at the 5′ endof SEQ ID NO:3, a poly(A) sequence before the first nucleotide at the 3′end of SEQ ID NO:4, N at the 3′ end of SEQ ID NO:1 represents 25nucleotides before the poly(A) sequence, N at the 5′ end of SEQ ID NO:2represents 27 nucleotides before the poly(T) sequence, there is apoly(T) sequence at the 5′ end of SEQ ID NO:3 after the final ‘N’ where‘N’ represents 27 nucleotides, there is a poly(A) sequence before thefirst ‘N’ at the 3′ end of SEQ ID NO: 4 where ‘N’ represents 25nucleotides.

However, the blunt-end ligation of Loop-1 linker(s) and DNA not only cangenerate the cleavable molecules (A), also generate other uncleavablemolecules (B and C) as well:

(B): (SEQ ID NO: 9)             (SEQ ID NO: 10)5′-GTCGTCN-----------------------NCTGCTG-3′3′-CAGCAGN-----------------------NGACGAC-5′ (SEQ ID NO:11)             (SEQ ID NO: 12)Where there is a poly(T) sequence after the final nucleotide at the 3′end of SEQ ID NO:9 wherein the ‘N’ represents 27 nucleotides, a poly(T)sequence before the first nucleotide at the 5′ end of SEQ ID NO: 10wherein ‘N’ represents 27 nucleotides, a poly(A) sequence after thefinal nucleotide at the 5′ end of SEQ ID NO:11 wherein ‘N’ represents 25nucleotides, a poly(A) sequence before the first nucleotide at the 3′end of SEQ ID NO:12 wherein ‘N’ represents 25 nucleotides.

(C): (SEQ ID NO:16)               (SEQ ID NO:13)5′-CAGCAGN-----------------------NGACGAC-3′3′-GTCGTCN-----------------------NCTGCTC-5′ (SEQ IDNO:14)               (SEQ ID NO:15)Where there is a poly(A) sequence after the final nucleotide at the 3′end of SEQ ID NO:16 wherein the ‘N’ represents 25 nucleotides, a poly(A)sequence before the first nucleotide at the 5′ end of SEQ ID NO:13wherein ‘N’ represents 25 nucleotides, a poly(T) sequence after thefinal nucleotide at the 5′ end of SEQ ID NO:14 wherein ‘N’ represents 25nucleotides, a poly(T) sequence before the first nucleotide at the 3′end of SEQ ID NO:15 wherein ‘N’ represents 27 nucleotides.

The present invention provides a single primer PCR amplification forselection of molecule (A) that can be cleaved. This single primer is aportion of sequences homolog to the strand with poly(A) stretch in theLoop-1 linker(s). The single primer PCR amplification completes aselection for molecule (A) that has two inversely orientated 5′-ends.The resulting PCR products are EcoP15I cleavable. The unclevablemolecules (B) and (C) are removed during the PCR process:

Wherein there is a poly(A) sequence after the final nucleotide at the 3′end of SEQ ID NO:16 wherein the ‘N’ represents 25 nucleotides, a poly(T)sequence before the first nucleotide at the 5′ end of SEQ ID NO:10wherein ‘N’ represents 27 nucleotides, a poly(T) sequence after thefinal nucleotide at the 5′ end of SEQ ID NO:14 wherein ‘N’ represents 25nucleotides, and a poly(A) sequence before the first nucleotide at the3′ end of SEQ ID NO:12 wherein ‘N’ represents 25 nucleotides.

3.) EcoP15I Digestion:

The type III restriction-modification enzyme-EcoP15I, consisting of twomodification (Mod) subunits and two restriction (Res) subunits, requiresthe interaction of two unmethylated, inversely oriented recognitionsites 5′-CAGCAG (SEQ ID NO:17) in head to head configuration to allow anefficient DNA cleavage. ATP is required for the cleavage. Cleavageefficiency is also affected by the distance between the two sites.EcoP15I can efficiently recognize the sites in distance up to 1.7 kb.

The Loop-1 linkers contain a type III restriction/modification enzymesite-EcoP15I and a type II restriction site-Fok1 adjacent to a PCRanchor. EcoP15I cleaves 25-27 bp outside of their recognition sequences,thus adjusting the number of the poly A/T sequences between EcoP151recognition site in Loop-1 linker(s) and DNA inserts, the resultingsiRNA polynucleotide pool has a distinct siRNA distribution infunctional length (e.g., 19-23 bp).

4) Blunt-ending and Loop-2 linker ligation: The DNA is cohesive-endedafter EcoP15I cleavage (sense strand 25 bp and antisense strand 27 bp),forming an over 5′ two bases hand and can be filled-in using a DNApolymerase in the presence of dNTPs at a final concentration is 0.02 mM.After a gel purification, the ligation of blunt-ended DNA and a Loop-2linker is catalyzed by T4 DNA ligase in the presence of ATP. Theligation products are template for the second PCR amplification.

5) Second PCR amplification: The blunt-end ligation of EcoP15I digestedsiRNAs and Loop-2 linker generates the following two molecules due todifferent orientations:

(A): Fok I (in Loop-2)   siRNA                 Fok I (in Loop-1)    (SEQID NO:18)                            (SEQ ID NO:19)5′-GGATG---(polyA)------------------(polyA)------GTAGG-3′3′-CCTAC---(polyT)------------------(polyT)------CATCC-5′    (SEQ IDNO:20)                            (SEQ ID NO:21) (B): Fok I (inLoop-1)   siRNA                 Fok I (in Loop-2)    (SEQ IDNO:18)                            (SEQ ID NO:22)5′-GGATG---(polyA)------------------(polyA)------CATCC-3′3′-CCTAC---(polyT)------------------(polyT)------GTAGG-5′    (SEQ IDNO:20)                            (SEQ ID NO:23)

After Fok I cleavage, only molecule (B) can generate the right cloningsites as below:

siRNA insert (SEQ ID NO:24) 5′AAAA-------------------------------3′ (SEQID NO:24) 3′------------------------------AAAA 5′

The second PCR amplification selects molecule (B) by 5′ Loop-1 and 3′Loop-2 primers:

6.) FokI Digestion:

Loop-2 linker plays a role in creating a PCR anchor and a cloning site.As a type II restriction enzyme-FokI can cleave any neighboring 9-13 bpsequences, after digestion, the DNA inserts generate over A₄ hands atboth 5′ ends as described in (5.).

7.) Cloning into an siRNA Expression Vector:

After a gel purification, the FokI digested products can be cloned intoa pre-prepared siRNA expression vector with over T₄ hands at both 3′ends (dephosphorylated), flanked by two tandem RNA polymerase IIIpromoters such as U6 and H1 (FIG. 2). Poly(A/T)₅ act as the initial andtermination signals for RNA polymerase III promoters as describedpreviously. After E. Coli transformation, the resulting siRNApolynucleotide pool has a size distribution same as that the Loop-1linkers restricted (e.g., 19-23 bp).

EXAMPLE 1 Oligo Sequences and Preparation

(A) Loop Phosphate Linkers: (Synthesized by Sigma-Aldrich)

Loop-1 Linker(s):

Each (100 mM) phosphate linker listed above was denatured at 95° C. in aPCR thermocycler for 1 minute (min) and then self annealed to a loop bycooling down to room temperature. After gel purification, each loopedlinker was diluted with 1×TE buffer to 50 mM and stored at −70° C. untiluse.

(B) PCR Primers (the Following PCR Primers Synthesized by Invitrogen)

PCR-1 primer: BH1: 5′ ACACATCCA ACGGATCCCAGTTCAG 3′ (SEQ ID NO:37) PCR-2primers: BH1: 5′ ACACATCCA ACGGATCCCAGTTCAG 3′ (SEQ ID NO:38) LG:5′ GACTCTGATGGATCGTCTGCAGAG 3′ (SEQ ID NO:39)

(C) PCR Quality Control Primers:

5′ U6: 5′ AAGGTCGGGCAGGAAGAGGGC 3′ (SEQ ID NO:40) 3′ H1:5′ TATTTGCATGTCGCTATGTGTTCT 3′ (SEQ ID NO:41)

Each one was diluted with 1×TE buffer to 10 mM and stored at −70° untiluse.

EXAMPLE 2 DNA Partial digestion by DNaseI

(1) Starting DNA: SARS coronavirus membrane protein (NCBI accession No.:AY536759) full-length cDNA (666 bp).

(2) One μg of SARS coronavirus membrane protein full-length cDNA waspartially digested into 100-300 bp blunt-end fragments by DNaseI (RocheBiosystem; 0.01˜0.03 U) in a Mn²⁺ buffer at a final concentration of 10mM Tris-HCl (pH9.0); 2 mM MgSO₄; 10 mM KCl; 8 mM (NH4))₂SO₄ and 1 mMMnCl₂. The reaction was performed on ice for 1 min following by heatinginactivation for 20 min at 70° C. The resulting products were analyzedon 1% Agarose gel.

EXAMPLE 3 Loop-1 Linkers Ligation and PCR-1 Amplification Loop-1 LinkersLigation

The partially digested products were ligated to Loop-1 phosphatelinker(s) (19-; 20-; 21-; 22- and 23-bp Loop-1 linker) respectively. 5 Lof ligation reaction mix contained: 2.5 μL partially digested DNA; 0.5μL 10×ligation reaction buffer (500 mM Tris-HCl (pH 7.5, 25° C.), 100 mMMgCl₂, 100 mM dithiothreitol (DTT), 25 μg/μL bovine serum albumin (BSA);1 μl (10 mM) Loop-1 linker; 0.5 μL 10 mM ATP, 0.5 μl T4 DNA Ligase(NEB). The reaction was performed at 16° C. overnight.

PCR-1 Amplification

The reaction mixture (50 μl) contained: 0.5 μl each Loop-1 ligatedproduct; 2 μL single primer BH1 (20 μM), 1 μl dNTP (10 mM), 5 μl 10×PCRbuffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl₂), 0.5 μl TaqDNA polymerase and 41 μL PCR H₂O. The thermal cycling was as follows:preheating at 95° C. for 1 min; 28 cycles of 95° C. for 15 s; 68° C. for1 min. Three μL of each PCR products were analyzed on a 1% Agarose gel.

EXAMPLE 4 EcoP15I Cleavage, Blunt-Ending and PAGE Purification EcoP15ICleavage

5 μL of each PCR-1 products generated by five (19-; 20-; 21-; 22- and 23bp) Loop-1 linkers were mixed. An EcoP15I digestion solution (100 μl)contained: 10 μL PCR-1 product mixture, 10 μL ATP (10 mM), 10 ul10×NEBuffer-3, 1 μL BSA (100×; 10 mg/mL), 10 μl (100 U) EcoP15I (NEB),59 μL D-H₂O and then placed in a 37° C. water bath for an overnightincubation.

Blunt-Ending

After phenol-chloroform extraction and ethanol precipitation, the pelletwas dissolved with 11 μL D-H₂O, add 1.5 μL (4.5 U) T4 DNA polymerase(NEB), 1.5 μL NEB Buffer-2 and 2 μL dNTP (1 mM) to a total 15 μLreaction volume and incubated at 37° C. for 15 min in a PCR thermalcycler. The reaction was inactivated by heating at 68° C. for 20 min.

PAGE Purification

1.5 μl DNA Loading Buffer (30 mM EDTA; 36% (V/V) glycerol; 0.05% (W/V)BPB, pH=7.0) was added into the 15 μL reaction and the reaction productwas loaded onto a polyacrylamide gel 6.5 μL/well for 20% TBEpolyacrylamide gel Electrophoresis (at 200V for 90 min). As shown inFIG. 6, the specific 66 bp DNA fragments were observed after 10% EtBrstaining (20 min) under a UV lamp. The gel was cut into piecescontaining 66 bp fragments, and put together in a fresh tube containing150 μL gel diffusion buffer (0.5M NH₄AC, 10 mM Mg(Ac)₂, 1 mM EDTA; pH8.0). The tube was incubated at 55° C. overnight. DNA was extracted withQIAEX II Gel Extraction Kit and elute DNA with D-H₂O.

EXAMPLE 5 Loop-2 Linker Ligation and PCR-2 Amplification Loop-2 LinkerLigation

Five μL ligation reaction contained: 2.5 μL DNA isolated by PAGE, 0.5 μL10×ligation reaction buffer (500 mM Tris-HCl (pH 7.5, 25° C.), 100 mMMgCl₂, 100 mM DTT, 25 μg/mL BSA), 1 μL (20 mM) Loop-2 linker, 0.5 μL ATP(10 mM), 0.5 μL T4 DNA Ligase (NEB) and 0.5 μL D-H₂O. The ligationreaction was incubated the reaction at 16° C. overnight in a PCR thermalcycler.

PCR-2 Amplification

The 5 μL ligation-2 product was diluted with 45 μL D-H₂O and take out0.5 μl as a template for PCR-2 amplification. A 50 μL reactioncontained: 0.5 μL diluted ligation-2 template, 5 μL 10×PCR buffer (200mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl₂), 1 μL LG primer (10 μM),1 μL BH1(10 μM), 1 μL dNTP (10 mM), 0.5 μL Taq DNA polymerase, 41 μL PCRH₂O. The thermal cycling was as follows: preheating at 95° C. for 1 min;28 cycles of 95° C. for 15 s; 68° C. for 1 min. After amplification, 10μL PCR product was analyzed on a 20% TBE polyacrylamide gel. A specific109 bp amplicon was observed after 10% EtBr staining (20 min) under a UVlamp. After phenol: chloroform extraction, the ethanol precipitation wasperformed at −20° C. at least for 2 hours. Pellet The DNA wascentrifuged and the resultant DNA pellet was dissolved in 20 μL D-H₂O.

EXAMPLE 6 FokI Digestion

A 100 μL reaction contained: 20 μL above PCR-2 product, 2 μL FokI (8 U;NEB), 10 μL NEBuffer-3 and 72 μL D-H₂O, which was incubated at 37° C.for 2 hrs. After the digestion reaction 10 μL of the Fok1 digested DNAwas analyzed on a 20% TBE polyacrylamide gel. The target bands weredistributed from 29 to 33 bp in length, which corresponds to siRNAhaving 19-23 bp having 10 bp cloning sites (“AAAAG” (SEQ ID NO:41) atboth 5′ ends). Three other 3 bands from top to bottom were: anundigested 109 bp band, a partial digested band (siRNA with a loop atone side), a Loop-1 and Loop-2 overlapping band (owing to Loop-1 andLoop-2 overlap each other, the signal is strong).

EXAMPLE 7 Vector Ligation and Completion of siRNA Library

Ligation into an siRNA Expression Vector

DNA fragments after FokI digestion were ligated into an siRNA expressionvector: pU6H1-GFP(NT Omics, USA) with double promoters of U6 and H1(FIG. 2). A 15 μL ligation mixture contained: 1 μL (100 ng) pU6H1-GFP, 2μL FokI digested DNA, 10 μL 1.5×Plasmid Ligation Buffer (90 mM Tris(PH8.5-9.0); 12 mM DTT; 60 mM MgCl₂; 40% PEG8000), 0.5 μL ATP (10 mM),0.25 μL T4 DNA Ligase (NEB), 2.25 μl D-H₂O. The reaction was incubatedat 16° C. for 30 min. 85 μL D-H₂O was added to the ligation mixture andethanol-precipitated an ethanol precipitated at −70° C. for 30 minutes.The precipitated DNA was centrifuged forming a DNA pellet and the pelletwas dissolved in 5 μL D-H₂O.

Electrotransformation

Electro-competent cells, MegaX DH10B™ (Invitrogen) were thawd on ice. 5μL ligation product was mixed in 40 μL electro-competent cells andplaced in ice for 1 min. The mixture was transferred into a cuvette(BioRad) to perform electrotransformation in a MicroPulser (BioRad).After transformation the mixture was transferred to a fresh tubecontaining 150 μL SOC medium (2% (W/V)) Tryptone, 0.5% (W/V) Yeast,0.05% (W/V) NaCl, 2.5 mM KCl, 11 mM MgCl₂, 20 mM glucose, pH=7.0) andincubated was performed at 37° C. with a continuous shaking for 40˜60min at 220 rpm, followed by plating the bacterial culture on a solidmedium (1% (W/V) Tryptone, 0.5% (W/V) NaCl, 1.5% (W/V) Agar, pH=7.0; 50μg/mL Kanamycine), which was incubated at 37° C. in an air incubatorovernight. One ligation reached 1×10³ independent clones (colonies).

EXAMPLE 8 siRNA Library Validatio Sample Inoculation

The well-isolated colonies were randomly picked and inoculated intoseparate 1.5 mL eppendorf tube (or 48-well plate), each containing 400μL LB medium (1% (W/V) Tryptone, 0.5% (W/V) Yeast, 1% (W/V) NaCl,pH=7.0). Incubation was performed at 37° C. with a continuous shakingfor 2 hours at 220 rpm.

PCR-Based Insert Screening

The reaction mixture (30 HL) contained: 2 μL above bacteria culture, 0.5μL 5′ U6 primer (10 μM), 0.5 μL 5′ H1 primer (10 μM), 0.5 μl dNTP (10mM), 3 L 10×PCR buffer (200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mMMgCl₂), 0.3 μL Taq DNA polymerase and 23.2 μL PCR H₂O. The thermalcycling was as follows: preheating at 95° C. for 5 min; 25 cycles of 95°C. for 15 s; 62° C. for 30 s; 72° C. for 40 s; holding at 72° C. for 7min for last extension. Five μL of each PCR products were analyzed on 1%Agarose gel. The positive bands of PCR products were ˜400 bp in length.The positive rate for sampling was 91.6% (44/48).

SfiI Digestion of PCR Products

The size of the siRNAs ranged from 19-23 bp in length. To furthervalidate siRNA positive clones, SfiI restriction enzyme was added to PCRproducts. The SfiI restriction site was originally in the MCS (multiplecloning sites) region of pU6/H1-GFP vector used. A positive digestionindicates siRNA insert negative ((just a vacant vector). The SfiIdigestion mixture contained: 4 μL above PCR products, 0.25 μL (5 U) SfiI(NEB), 0.75 μL NEBuffer-2. Digestion was incubated at 50° C. for 1.5 hrin a PCR thermal cycler. Five μL SfiI digestion mixture was analyzed on1% Agarose gel. Out of the 43 testing samples, only 1 could be digestedby SfiI, indicating that the recombination rate of the experimental SARSsiRNA library was 97.6% (43/44).

Plasmid Mini Preparation and Sequencing

100 μL of bacteria culture was inoculated with PCR/SfiI positiveconstructs into a culture tube containing 4 mL LB medium (1% (W/V)Tryptone, 0.5% (W/V) Yeast, 1% (W/V) NaCl, pH=7.0; 50 μL/mL Kanamycine).An overnight incubation was performed at 37° with a continuous shakingfor 2 hours at 220 rpm. 3.2 mL of it was taken for mini sale plasmidextraction with TIAN Prep Mini plasmid Kit ((TIANGEN), store theremaining in 20% glycerol at −20% for further use. One μL (150˜200 ng)purified plasmid of each sample was analyzed on 1% Agarose gel. Theplasmids were sequenced (Shanghai Invitrogen) and the resultant iNAsequences were aligned with the SARS full-length cDNA sequence by analignment program software. The results are listed in

TABLE I The length of siRNA clones is distributed from 19 to 23 bp witha random binding sites distribution. The results confirm that theexperimental SRAR siRNA library is representative both in length andbinding sites. TABLE I. Sequencing Results for Clones Selected from theExperimental SARS siRNA Library Length Sites NO. Clone NO. siRNAsequences (bp) (nt)  1 S061218-1 TACAATTTGCCTATTCTAATC 21 101~121 (SEQID NO:43)  2 S061218-11 GGCTCTTGTGGCCAGTAACA 20 161~181 (SEQ ID NO:44) 3 S061220-10 GGAAAACAAGCTTTATTATG 20 529~510 (SEQ ID NO:45)  4S061220-19 TACGGTAGCGGTTGTATGC 19  87~69 (SEQ ID NO:46)  5 S070115-21TATTCTAATCGGAACAGGTT 20 112~131 (SEQ ID NO:47)  6 S070115-26GAGCAAACAGCCTGAAGGAAGC 22 378~357 (SEQ ID NO:48)  7 S070115-46GTACCCGCTCAATGTGGTCA 20 311~330 (SEQ ID NO:49)  8 S070119-27GTACATAATAAAGCTTGTTTTCC 23 135~157 (SEQ ID NO:50)  9 S070119-28AGAATGTTTGTTTCTGGGT 19 332~312 (SEQ ID NO:51) 10 S070119-30CATTGGTGCTGTGATCATTC 20 414~433 (SEQ ID NO:52) 11 S070119-31GAGAATGTTTGTTTCTGGGT 20 332~314 (SEQ ID NO:53) 12 S070119-32CTTTATTATGTACAAAAACC 20 539~520* (SEQ ID NO:54) 13 S070119-34GATTAGAATAGGCAAATTGT 55 20 565~546 14 S070122-7 GGAAGCAACGAAGTAGCTAAGCC56 23 395~373 15 S070122-12 GAGCGGGTACGAGCAAACAGCC 22 368~347 (SEQ IDNO:57) 16 S070122-14 TCTCCGGGGGACAATTGTGAC 21 366~386* (SEQ ID NO:58) 17S070122-19 TGTTACTACAATTTGCCTATTC 22  95~116 (SEQ ID NO:59) 18S070122-22 GCTTTATTATGTACAAAAACC 21 539~519* (SEQ ID NO:60) 19S070122-30 GAATGACCACATTGAGCGGGT 21 354~334 (SEQ ID NO:61) 20 S070122-32GTGATGTAGCCACAGTGATC 20 169~150 (SEQ ID NO:62) 21 S070122-48TCAACCCAGAAACAAAGATTC 21 332~352 (SEQ ID NO:63) 22 S070307-4GTATTGTAGGCTTGATGTGGCT 22 254~275* (SEQ ID NO:64) 23 S070307-45CTACAATTTGCCTATTCTAATC 22 100~121 (SEQ ID NO:65) 24 S070307-48TACAATACAAGCCATTGCAATC 22 427~406 (SEQ ID NO:66) 25 S070316-11CATCAAGCCTACAATACAAGCC 22 418~397* (SEQ ID NO:67) (* Repeated clones)

1. A method for producing a pool of interfering nucleic acid (iNA)polynucleotides comprising the following steps: a) partially digesting aDNA producing DNA constructs; b) ligating Loop-1 linkers to one end ofthe DNA constructs wherein the loop-1 linker contains a type IIrestriction enzyme recognition site and a type III restriction enzymerecognition site; c) cleaving the DNA constructs with a type IIIrestriction/modification enzyme; d) ligating a Loop-2 linker onto an endof each of the DNA constructs, wherein said Loop-2 linker has a type IIrestriction enzyme cognition site producing DNA constructs that have aLoop-1 linker at one end and a Loop-2 linker at a second end of the DNAconstruct; e) digesting the DNA constructs with a type II restrictionenzyme; f) ligating the DNA constructs into iNA expression vectors; andg) transfecting the transcription vectors into cells under conditionswherein iNAs are produced.
 2. The method of claim one wherein the RNAexpression vectors have one or more RNA polymerase III promoters.
 3. Themethod of claim 2 wherein the RNA expression vectors have two RNApolymerase III promoters, wherein a DNA construct that is to betranscribed into RNA is ligated into the vector between the twopromoters.
 4. The method of claim 3 wherein one of the promoters is a U6promoter and one of the promoters is a H1 promoter.
 5. The method ofclaim 1 wherein a termination signal is attached to each end of the DNAconstruct.
 6. The method of claim 5 wherein the termination signals arepolyA/polyT construct.
 7. The method of claim 1 wherein the DNAconstruct of step (d) is amplified.
 8. The method of claim 7 wherein theDNA construct is amplified by a polymerase chain reaction (PCR).
 9. ADNA construct for producing a iNA comprised of a first end, a second endand a middle DNA sequence in between the first in second ends whereinthe first end is a loop-1 linker DNA sequence and the second end of theDNA construct is a loop-2 linker DNA sequence, wherein the middle DNAsequence encodes a iNA sequence having a length of from 16 to 27 bps,wherein the loop-1 linker has a type II restriction enzyme recognitionsite and a type III restriction enzyme site, and wherein the loop-2linker DNA sequence has a type II restriction enzyme recognition site.10. The DNA construct of claim 9 wherein at each end of the DNAconstruct is a termination signal for RNA polymerase III.
 11. The DNAconstruct of claim 10 wherein the termination signal sequence is apolyA/polyT construct.
 12. The DNA construct of claim 11 wherein thepolyA sequence has 4 to 7 ‘A’s and the polyT has 4 to 7 ‘T’s.
 13. A iNAexpression vector for producing a iNA comprised of two RNA polymeraseIII promoters and a DNA construct encoding an iNA, wherein the DNAconstruct has a first end and a second end, wherein the DNA construct isplaced between the two promoters, wherein a RNA polymerase IIItermination signal sequence is ligated onto each end of the DNAconstruct and wherein the DNA is a sequence having 16 to 27 base-pairsof nucleotides.
 14. The expression vector of claim 13 wherein the RNApolymerase III promoters are either a U6 RNA polymerase III promoter ora H1 RNA polymerase III promoter.
 15. The expression vector of constructof claim 13 wherein the termination signal is a polyA/polyT construct.